Branched-Chain Amino Acids Deficiency Promotes Diabetic Neuropathic Pain Through Upregulating LAT1 and Inhibiting Kv1.2 Channel.

Diabetic neuropathic pain (DNP), one of the most common complications of diabetes, is characterized by bilateral symmetrical distal limb pain and substantial morbidity. To compare the differences  is aimed at serum metabolite levels between 81 DNP and 73 T2DM patients without neuropathy and found that the levels of branched-chain amino acids (BCAA) are significantly lower in DNP patients than in T2DM patients. In high-fat diet/low-dose streptozotocin (HFD/STZ)-induced T2DM and leptin receptor-deficient diabetic (db/db) mouse models, it is verified that BCAA deficiency aggravated, whereas BCAA supplementation alleviated DNP symptoms. Mechanistically, using a combination of RNA sequencing of mouse dorsal root ganglion (DRG) tissues and label-free quantitative proteomic analysis of cultured cells, it is found that BCAA deficiency activated the expression of L-type amino acid transporter 1 (LAT1) through ATF4, which is reversed by BCAA supplementation. Abnormally upregulated LAT1 reduced Kv1.2 localization to the cell membrane, and inhibited Kv1.2 channels, thereby increasing neuronal excitability and causing neuropathy. Furthermore, intraperitoneal injection of the LAT1 inhibitor, BCH, alleviated DNP symptoms in mice, confirming that BCAA-deficiency-induced LAT1 activation contributes to the onset of DNP. These findings provide fresh insights into the metabolic differences between DNP and T2DM, and the development of approaches for the management of DNP.

m6A epitranscriptomic modification of inflammation in cardiovascular disease.

Cardiovascular disease is currently the number one cause of death endangering human health. There is currently a large body of research showing that the development of cardiovascular disease and its complications is often accompanied by inflammatory processes. In recent years, epitranscriptional modifications have been shown to be involved in regulating the pathophysiological development of inflammation in cardiovascular diseases, with 6-methyladenine being one of the most common RNA transcriptional modifications. In this review, we link different cardiovascular diseases, including atherosclerosis, heart failure, myocardial infarction, and myocardial ischemia-reperfusion, with inflammation and describe the regulatory processes involved in RNA methylation. Advances in RNA methylation research have revealed the close relationship between the regulation of transcriptome modifications and inflammation in cardiovascular diseases and brought potential therapeutic targets for disease diagnosis and treatment. At the same time, we also discussed different cell aspects. In addition, in the article we also describe the different application aspects and clinical pathways of RNA methylation therapy. In summary, this article reviews the mechanism, regulation and disease treatment effects of m6A modification on inflammation and inflammatory cells in cardiovascular diseases in recent years. We will discuss issues facing the field and new opportunities that may be the focus of future research.

Redox homeostasis in cardiac fibrosis: Focus on metal ion metabolism.

Cardiac fibrosis is a major public health problem worldwide, with high morbidity and mortality, affecting almost all patients with heart disease worldwide. It is characterized by fibroblast activation, abnormal proliferation, excessive deposition, and abnormal distribution of extracellular matrix (ECM) proteins. The maladaptive process of cardiac fibrosis is complex and often involves multiple mechanisms. With the increasing research on cardiac fibrosis, redox has been recognized as an important part of cardiac remodeling, and an imbalance in redox homeostasis can adversely affect the function and structure of the heart. The metabolism of metal ions is essential for life, and abnormal metabolism of metal ions in cells can impair a variety of biochemical processes, especially redox. However, current research on metal ion metabolism is still very limited. This review comprehensively examines the effects of metal ion (iron, copper, calcium, and zinc) metabolism-mediated redox homeostasis on cardiac fibrosis, outlines possible therapeutic interventions, and addresses ongoing challenges in this rapidly evolving field.

High paternal homocysteine causes ventricular septal defects in mouse offspring.

Maternal hyperhomocysteinemia is widely considered as an independent risk of congenital heart disease (CHD). However, whether high paternal homocysteine causes CHD remains unknown. Here, we showed that increased homocysteine levels of male mice caused decreased sperm count, sperm motility defect and ventricular septal defect of the offspring. Moreover, high levels of paternal homocysteine decrease sperm DNMT3A/3B, accompanied with changes in DNA methylation levels in the promoter regions of CHD-related genes. Folic acid supplement could decrease the occurrence of VSD in high homocysteine male mice. This study reveals that increased paternal homocysteine level increases VSD risk in the offspring, indicating that decreasing paternal homocysteine may be an intervening target of CHD.

Ketogenic diet-produced ß-hydroxybutyric acid accumulates brain GABA and increases GABA/glutamate ratio to inhibit epilepsy.

Ketogenic diet (KD) alleviates refractory epilepsy and reduces seizures in children. However, the metabolic/cell biologic mechanisms by which the KD exerts its antiepileptic efficacy remain elusive. Herein, we report that KD-produced ß-hydroxybutyric acid (BHB) augments brain gamma-aminobutyric acid (GABA) and the GABA/glutamate ratio to inhibit epilepsy. The KD ameliorated pentetrazol-induced epilepsy in mice. Mechanistically, KD-produced BHB, but not other ketone bodies, inhibited HDAC1/HDAC2, increased H3K27 acetylation, and transcriptionally upregulated SIRT4 and glutamate decarboxylase 1 (GAD1). BHB-induced SIRT4 de-carbamylated and inactivated glutamate dehydrogenase to preserve glutamate for GABA synthesis, and GAD1 upregulation increased mouse brain GABA/glutamate ratio to inhibit neuron excitation. BHB administration in mice inhibited epilepsy induced by pentetrazol. BHB-mediated relief of epilepsy required high GABA level and GABA/glutamate ratio. These results identified BHB as the major antiepileptic metabolite of the KD and suggested that BHB may serve as an alternative and less toxic antiepileptic agent than KD.

Epigenetic signatures in cardiac fibrosis: Focusing on noncoding RNA regulators as the gatekeepers of cardiac fibroblast identity.

Cardiac fibroblasts play a pivotal role in cardiac fibrosis by transformation of fibroblasts into myofibroblasts, which synthesis and secrete a large number of extracellular matrix proteins. Ultimately, this will lead to cardiac wall stiffness and impaired cardiac performance. The epigenetic regulation and fate reprogramming of cardiac fibroblasts has been advanced considerably in recent decades. Non coding RNAs (microRNAs, lncRNAs, circRNAs) regulate the functions and behaviors of cardiac fibroblasts, including proliferation, migration, phenotypic transformation, inflammation, pyroptosis, apoptosis, autophagy, which can provide the basis for novel targeted therapeutic treatments that abrogate activation and inflammation of cardiac fibroblasts, induce different death pathways in cardiac fibroblasts, or make it sensitive to established pathogenic cells targeted cytotoxic agents and biotherapy. This review summarizes our current knowledge in this field of ncRNAs function in epigenetic regulation and fate determination of cardiac fibroblasts as well as the details of signaling pathways contribute to cardiac fibrosis. Moreover, we will comment on the emerging landscape of lncRNAs and circRNAs function in regulating signal transduction pathways, gene translation processes and post-translational regulation of gene expression in cardiac fibroblast. In the end, the prospect of cardiac fibroblasts targeted therapy for cardiac fibrosis based on ncRNAs is discussed.

Lipid metabolism reprogramming in cardiac fibrosis.

Cardiac fibrosis is a critical pathophysiological process that occurs with diverse types of cardiac injury. Lipids are the most important bioenergy substrates for maintaining optimal heart performance and act as second messengers to transduce signals within cardiac cells. However, lipid metabolism reprogramming is a double-edged sword in the regulation of cardiomyocyte homeostasis and heart function. Moreover, lipids can exert diverse effects on cardiac fibrosis through different signaling pathways. In this review, we provide a brief overview of aberrant cardiac lipid metabolism and recent progress in pharmacological research targeting lipid metabolism alterations in cardiac fibrosis.

Clinical phenotypic and genotypic analysis of 5 pediatric patients with β-ketothiolase deficiency / 中华儿科杂志

Objective: To summarize the clinical and genetic characteristics of children with β-ketothiolase deficiency (BKTD). Methods: The clinical characteristics, biochemical, markers detected by tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC/MS), as well as the variants in ACAT1 gene among 5 children with BKTD in Children's Hospital of Chongqing Medical University between October 2018 and December 2022 were retrospectively analyzed. Results: The onset age of the disease in 5 patients (4 males and 1 female) ranged from 9.7 to 28.0 months. During the acute phase, severe metabolic acidosis was observed with a pH of 6.9-7.1, as well as hypoglycaemia (2.3-3.4 mmol/L) and positive urinary ketone bodies (+-++++). Blood levels of methylcrotonyl carnitine, methylmalonyl carnitine and malonyl carnitine were 0.03-0.42, 0.34-1.43 and 0.83-3.53 μmol/L respectively and were significantly elevated. Urinary 2-methyl-3-hydroxybutyric acid was 22-202 and 3-hydroxybutyric acid was 4-6 066, both were higher than the normal levels. Methylcrotonylglycine was mild elevated (0-29). The metabolites detected by MS/MS and GC/MS were significantly reduced after treatment. Analysis of ACAT1 gene mutation was performed in 5 children. Most variants were missense (8/9). Four previously unreported variants were identified: c.678G>T (p.Trp226Cys), c.302A>G (p.Gln101Arg), c.627_629dupTGA (p.Asn209_Glu210insAsp) and c.316C>T (p.Gln106Ter), the first 2 variants were predicted to be damaging by SIFT, PolyPhen-2 and Mutation Taster software. c.316C>T (p.Gln106Ter) is a nonsense variant. Conclusions: β-ketothiolase deficiency is relatively rare, lacks specific clinical manifestations, however severe metabolic acidosis, hypoglycemia, and ketosis during the acute onset were consistent findings. Missense mutations in the ACAT1 gene are common genetic causes of β-ketothiolase deficiency.

Formate Might Be a Novel Potential Serum Metabolic Biomarker for Type 2 Diabetic Peripheral Neuropathy.

Background: As one of the most frequent complications of type 2 diabetes mellitus (T2DM), diabetic peripheral neuropathy (DPN) shows a profound impact on 50% of patients with symptoms of neuropathic pain, numbness and other paresthesia. No valid serum biomarkers for the prediction of DPN have been identified in the clinic so far. This study is to investigate the potential serum biomarkers for DPN firstly based on 1H-Nuclear Magnetic Resonance (1H-NMR)-based metabolomics technique. Methods: Thirty-six patients enrolled in this study were divided into two groups: 18 T2DM patients without DPN (T2DM group) and 18 T2DM patients with DPN (DPN group). Serum metabolites were measured via 1H-NMR spectroscopy. Bioinformatic approaches including principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), independent sample t-test, Fisher's test, Pearson and Spearman correlation analysis, Stepwise multiple linear regression analysis and receiver operating characteristic (ROC) curve analysis were used to identify the potential altered serum biomarkers. Results: A total of 20 metabolites were obtained and further analyzed. Formate was identified as the only potential biomarker that decreased in the DPN group with statistical significance after multiple comparisons (p<0.05). Formate also displayed a negative relationship with some elevated clinical markers in DPN. ROC curve analysis showed a good discriminative ability for formate in DPN with an area under the curve (AUC) value of 0.981. Conclusion: Formate could be considered a potential serum metabolic biomarker for DPN. The reduced level of formate in DPN may be associated with mitochondrial dysfunction and gut microbiota alteration. Monitoring the level of serum formate would be an important strategy for the early diagnosis of DPN and a supplement of formate may be a promising treatment for DPN in the future.

Proteome profiling of early gestational plasma reveals novel biomarkers of congenital heart disease.

Prenatal diagnosis of congenital heart disease (CHD) relies primarily on fetal echocardiography conducted at mid-gestational age-the sensitivity of which varies among centers and practitioners. An objective method for early diagnosis is needed. Here, we conducted a case-control study recruiting 103 pregnant women with healthy offspring and 104 cases with CHD offspring, including VSD (42/104), ASD (20/104), and other CHD phenotypes. Plasma was collected during the first trimester and proteomic analysis was performed. Principal component analysis revealed considerable differences between the controls and the CHDs. Among the significantly altered proteins, 25 upregulated proteins in CHDs were enriched in amino acid metabolism, extracellular matrix receptor, and actin skeleton regulation, whereas 49 downregulated proteins were enriched in carbohydrate metabolism, cardiac muscle contraction, and cardiomyopathy. The machine learning model reached an area under the curve of 0.964 and was highly accurate in recognizing CHDs. This study provides a highly valuable proteomics resource to better recognize the cause of CHD and has developed a reliable objective method for the early recognition of CHD, facilitating early intervention and better prognosis.

WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation.

Dysregulated lipid metabolism occurs in pathological processes characterized by cell proliferation and migration. Nonetheless, the mechanism of increased mitochondrial lipid oxidation is poorly appreciated in diabetic cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. Herein, increased WTAP expression promotes cardiac fibroblast proliferation and migration, contributing to diabetic cardiac fibrosis. Knockdown of WTAP suppresses mitochondrial lipid oxidation, fibroblast proliferation and migration to ameliorate diabetic cardiac fibrosis. Mechanistically, WTAP-mediated m6A methylation of AR induced its degradation, dependent on YTHDF2. Additionally, AR directly interacts with mitochondrial lipid oxidation enzyme Decr1; overexpression of AR-suppressed Decr1-mediates mitochondrial lipid oxidation, inhibiting cardiac fibroblast proliferation and migration. Knockdown of AR produced the opposite effect. Clinically, increased WTAP and YTHDF2 levels correlate with decreased AR expression in human DCM heart tissue. We describe a mechanism wherein WTAP boosts higher mitochondrial lipid oxidation, cardiac fibroblast proliferation, and migration by enhancing AR methylation in a YTHDF2-dependent manner.

Proteogenomics of clear cell renal cell carcinoma response to tyrosine kinase inhibitor.

The tyrosine kinase inhibitor (TKI) Sunitinib is one the therapies approved for advanced renal cell carcinoma. Here, we undertake proteogenomic profiling of 115 tumors from patients with clear cell renal cell carcinoma (ccRCC) undergoing Sunitinib treatment and reveal the molecular basis of differential clinical outcomes with TKI therapy. We find that chromosome 7q gain-induced mTOR signaling activation is associated with poor therapeutic outcomes with Sunitinib treatment, whereas the aristolochic acid signature and VHL mutation synergistically caused enhanced glycolysis is correlated with better prognosis. The proteomic and phosphoproteomic analysis further highlights the responsibility of mTOR signaling for non-response to Sunitinib. Immune landscape characterization reveals diverse tumor microenvironment subsets in ccRCC. Finally, we construct a multi-omics classifier that can detect responder and non-responder patients (receiver operating characteristic-area under the curve, 0.98). Our study highlights associations between ccRCC molecular characteristics and the response to TKI, which can facilitate future improvement of therapeutic responses.

Crosstalk between oxidative stress and epigenetic marks: New roles and therapeutic implications in cardiac fibrosis.

With the in-depth investigation of cardiac fibrosis, oxidative stress (OS) has been recognized as a significant pathophysiological pathway involved in cardiac remodeling and progression. OS is a condition characterized by the disruption of equilibrium between reactive oxygen species (ROS) produced by the organism and the antioxidant defense system, resulting in adverse effects on the structure and function of the heart. The accumulation of reactive substances beyond cellular thresholds disrupts the normal physiology of both cardiomyocytes and non-cardiomyocytes, leading to OS, inflammation, hypertrophy, and cardiac fibrosis. Furthermore, cardiac OS also modulates several crucial genes involved in maintaining cellular homeostasis, including those associated with mitochondrial biogenesis, injury, and antioxidant defense, which are inevitably associated with concurrent epigenetic changes. Multiple studies have demonstrated the crucial role of epigenetic modifications in regulating cardiac OS. Consequently, modulating OS through targeted epigenetic modifications emerges as a potentially promising therapeutic strategy for managing cardiac fibrosis. This article provides a new review of current research on this subject and proposes that epigenetics may improve OS-induced cardiac fibrosis.

Glycolytic reprogramming in organ fibrosis: New dynamics of the epigenetic landscape.

Accumulating evidence has shown that aerobic glycolysis is essential for the establishment and maintenance of the fibrotic phenotype, so treatments targeting glycolytic reprogramming may become an important strategy to reduce fibrosis. Here, we reviewed current evidence on the glycolytic reprogramming in organ fibrosis, new dynamics of the epigenetic landscape. Epigenetic regulation of the expression of specific genes involved mediates glycolytic reprogramming, thereby affecting fibrosis progression. A comprehensive understanding of the interplay between aerobic glycolysis and epigenetics holds great promise for the treatment and intervention of fibrotic diseases. This article aims to comprehensively review the effect of aerobic glycolysis on organ fibrosis, and to elucidate the relevant epigenetic mechanisms of glycolytic reprogramming in different organs.

METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation.

Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.

Mitochondrial quality control in cardiac fibrosis: Epigenetic mechanisms and therapeutic strategies.

Cardiac fibrosis (CF) is considered an ultimate common pathway of a wide variety of heart diseases in response to diverse pathological and pathophysiological stimuli. Mitochondria are characterized as isolated organelles with a double-membrane structure, and they primarily contribute to and maintain highly dynamic energy and metabolic networks whose distribution and structure exert potent support for cellular properties and performance. Because the myocardium is a highly oxidative tissue with high energy demands to continuously pump blood, mitochondria are the most abundant organelles within mature cardiomyocytes, accounting for up to one-third of the total cell volume, and play an essential role in maintaining optimal performance of the heart. Mitochondrial quality control (MQC), including mitochondrial fusion, fission, mitophagy, mitochondrial biogenesis, and mitochondrial metabolism and biosynthesis, is crucial machinery that modulates cardiac cells and heart function by maintaining and regulating the morphological structure, function and lifespan of mitochondria. Certain investigations have focused on mitochondrial dynamics, including manipulating and maintaining the dynamic balance of energy demand and nutrient supply, and the resultant findings suggest that changes in mitochondrial morphology and function may contribute to bioenergetic adaptation during cardiac fibrosis and pathological remodeling. In this review, we discuss the function of epigenetic regulation and molecular mechanisms of MQC in the pathogenesis of CF and provide evidence for targeting MQC for CF. Finally, we discuss how these findings can be applied to improve the treatment and prevention of CF.

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. In vivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

Amino acids downregulate SIRT4 to detoxify ammonia through the urea cycle.

Ammonia production via glutamate dehydrogenase is inhibited by SIRT4, a sirtuin that displays both amidase and non-amidase activities. The processes underlying the regulation of ammonia removal by amino acids remain unclear. Here, we report that SIRT4 acts as a decarbamylase that responds to amino acid sufficiency and regulates ammonia removal. Amino acids promote lysine 307 carbamylation (OTCCP-K307) of ornithine transcarbamylase (OTC), which activates OTC and the urea cycle. Proteomic and interactome screening identified OTC as a substrate of SIRT4. SIRT4 decarbamylates OTCCP-K307 and inactivates OTC in an NAD+-dependent manner. SIRT4 expression was transcriptionally upregulated by the amino acid insufficiency-activated GCN2-eIF2α-ATF4 axis. SIRT4 knockout in cultured cells caused higher OTCCP-K307 levels, activated OTC, elevated urea cycle intermediates and urea production via amino acid catabolism. Sirt4 ablation decreased male mouse blood ammonia levels and ameliorated CCl4-induced hepatic encephalopathy phenotypes. We reveal that SIRT4 safeguards cellular ammonia toxicity during amino acid catabolism.

Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response.

Dysregulated amino acid increases the risk for heart failure (HF) via unclear mechanisms. Here, we find that increased plasma tyrosine and phenylalanine levels are associated with HF. Increasing tyrosine or phenylalanine by high-tyrosine or high-phenylalanine chow feeding exacerbates HF phenotypes in transverse aortic constriction and isoproterenol infusion mice models. Knocking down phenylalanine dehydrogenase abolishes the effect of phenylalanine, indicating that phenylalanine functions by converting to tyrosine. Mechanistically, tyrosyl-tRNA synthetase (YARS) binds to ataxia telangiectasia and Rad3-related gene (ATR), catalyzes lysine tyrosylation (K-Tyr) of ATR, and activates the DNA damage response (DDR) in the nucleus. Increased tyrosine inhibits the nuclear localization of YARS, inhibits the ATR-mediated DDR, accumulates DNA damage, and elevates cardiomyocyte apoptosis. Enhancing ATR K-Tyr by overexpressing YARS, restricting tyrosine, or supplementing tyrosinol, a structural analog of tyrosine, promotes YARS nuclear localization and alleviates HF in mice. Our findings implicate facilitating YARS nuclear translocation as a potential preventive and/or interfering measure against HF.